Target enrichment and high‐throughput sequencing of 80 ribosomal protein genes to identify mutations associated with Diamond‐Blackfan anaemia

Diamond‐Blackfan anaemia (DBA) is caused by inactivating mutations in ribosomal protein (RP) genes, with mutations in 13 of the 80 RP genes accounting for 50–60% of cases. The remaining 40–50% cases may harbour mutations in one of the remaining RP genes, but the very low frequencies render conventional genetic screening as challenging. We, therefore, applied custom enrichment technology combined with high‐throughput sequencing to screen all 80 RP genes. Using this approach, we identified and validated inactivating mutations in 15/17 (88%) DBA patients. Target enrichment combined with high‐throughput sequencing is a robust and improved methodology for the genetic diagnosis of DBA.     

Diagnosing and treating Diamond Blackfan anaemia: results of an international clinical consensus conference

Diamond Blackfan anaemia (DBA) is a rare, genetically and clinically heterogeneous, inherited red cell aplasia. Classical DBA affects about seven per million live births and presents during the first year of life. However, as mutated genes have been discovered in DBA, non-classical cases with less distinct phenotypes are being described in adults as well as children. In caring for these patients it is often difficult to have a clear understanding of the treatment options and their outcomes because of the lack of complete information on the natural history of the disease. The purpose of this document is to review the criteria for diagnosis, evaluate the available treatment options, including corticosteroid and transfusion therapies and stem cell transplantation, and propose a plan for optimizing patient care. Congenital anomalies, mode of inheritance, cancer predisposition, and pregnancy in DBA are also reviewed. Evidence-based conclusions will be made when possible; however, as in many rare diseases, the data are often anecdotal and the recommendations are based upon the best judgment of experienced clinicians. The recommendations regarding the diagnosis and management described in this report are the result of deliberations and discussions at an international consensus conference.     

Ribosomal RNA analysis in the diagnosis of Diamond‐Blackfan Anaemia

Diamond‐Blackfan anaemia (DBA) is an inherited disease characterized by pure erythroid aplasia that has been tagged as a ‘ribosomopathy’. We report a multi‐centre study focused on the analysis of rRNA processing of 53 Italian DBA patients using capillary electrophoresis analysis of rRNA maturation of the 40S and 60S ribosomal subunits. The ratio of 28S/18S rRNA was higher in patients with mutated ribosomal proteins (RPs) of the small ribosomal subunit. In contrast, patients with mutated RPs of the large ribosomal subunit (RPLs) had a lower 28S/18S ratio. The assay reported here would be amenable for development as a diagnostic tool.     

Glucocorticoids improve erythroid progenitor maintenance and dampen Trp53 response in a mouse model of Diamond–Blackfan anaemia

Diamond–Blackfan anaemia (DBA) is a rare congenital disease causing severe anaemia and progressive bone marrow failure. The majority of patients carry mutations in ribosomal proteins, which leads to depletion of erythroid progenitors in the bone marrow. As many as 40% of all DBA patients receive glucocorticoids to alleviate their anaemia. However, despite their use in DBA treatment for more than half a century, the therapeutic mechanisms of glucocorticoids remain largely unknown. Therefore we sought to study disease specific effects of glucocorticoid treatment using a ribosomal protein s19 (Rps19) deficient mouse model of DBA. This study determines for the first time that a mouse model of DBA can respond to glucocorticoid treatment, similar to DBA patients. Our results demonstrate that glucocorticoid treatment reduces apoptosis, rescues erythroid progenitor depletion and premature differentiation of erythroid cells. Furthermore, glucocorticoids prevent Trp53 activation in Rps19‐deficient cells‐ in a disease‐specific manner. Dissecting the therapeutic mechanisms behind glucocorticoid treatment of DBA provides indispensible insight into DBA pathogenesis. Identifying mechanisms important for DBA treatment also enables development of more disease‐specific treatments of DBA.     

Mesenchymal stromal cells from Shwachman‐Diamond syndrome patients fail to recreate a bone marrow niche in vivo and exhibit impaired angiogenesis

Shwachman‐Diamond syndrome (SDS) is a rare multi‐organ recessive disease mainly characterised by pancreatic insufficiency, skeletal defects, short stature and bone marrow failure (BMF). As in many other BMF syndromes, SDS patients are predisposed to develop a number of haematopoietic malignancies, particularly myelodysplastic syndrome and acute myeloid leukaemia. However, the mechanism of cancer predisposition in SDS patients is only partially understood. In light of the emerging role of mesenchymal stromal cells (MSCs) in the regulation of bone marrow homeostasis, we assessed the ability of MSCs derived from SDS patients (SDS‐MSCs) to recreate a functional bone marrow niche, taking advantage of a murine heterotopic MSC transplant model. We show that the ability of semi‐cartilaginous pellets (SCPs) derived from SDS‐MSCs to generate complete heterotopic ossicles in vivo is severely impaired in comparison with HD‐MSC‐derived SCPs. Specifically, after in vitro angiogenic stimuli, SDS‐MSCs showed a defective ability to form correct networks, capillary tubes and vessels and displayed a marked decrease in VEGFA expression. Altogether, these findings unveil a novel mechanism of SDS‐mediated haematopoietic dysfunction based on hampered ability of SDS‐MSCs to support angiogenesis. Overall, MSCs could represent a new appealing therapeutic target to treat dysfunctional haematopoiesis in paediatric SDS patients.     

Egyptian tale from India: application of whole‐exome sequencing in diagnosis of atypical familial Mediterranean fever

Clinical diagnosis of autoinflammatory diseases requires a high degree of clinical suspicion and clinching molecular evidence to substantiate the diagnosis. This is more so in populations with low prevalence of these disorders. In this report, we describe the case of a young man from India with recurrent fever and persistent arthritis. The patient's forefathers were of Egyptian ancestry who practiced consanguinity. Molecular genetic analysis using whole‐exome sequencing suggested the presence of variants c.443A>T:p.E148V and c.442G>C:p.E148Q in the MEFV gene, earlier independently shown to be associated with familial Mediterranean fever (FMF) in a compound heterozygous state. The variants were further confirmed by capillary sequencing. This report also highlights the application of whole exome sequencing to delineate the allelic differences in the variants apart from serving as a quick genetic screening approach for autoinflammatory diseases. To the best of our knowledge, this is the first report of a compound heterozygosity for the two well‐characterized variants associated with atypical FMF in a patient.     

Efficacy and safety of romiplostim in refractory aplastic anaemia: a Phase II/III,multicentre, open‐label study

A previous dose‐finding study has suggested that romiplostim is effective in patients with refractory aplastic anaemia (AA) and 10 µg/kg once weekly was recommended as a starting dose. In this Phase II/III, multicentre, open‐label study, romiplostim was administered subcutaneously at a fixed dose of 10 µg/kg once weekly for 4 weeks (weeks 1–4) followed by weekly doses (5, 10, 15 and 20 µg/kg) titrated by platelet response for up to 52 weeks (weeks 5–52). A total of 31 patients with AA who were refractory to immunosuppressive therapy (IST) and thrombocytopenia (platelet count of ≤30 × 10⁹/l) were enrolled. The primary efficacy endpoint of the proportion of patients achieving any haematological (platelet, neutrophil and erythrocyte) response at week 27 was 84% [95% confidence interval (CI) 66–95%]. Trilineage response was 39% (95% CI 22–58%) at week 53. The most common treatment‐related adverse events (AEs) were headache and muscle spasms (each 13%). All AEs were mild or moderate except for three patients with Grade 3 hepatic AEs; no AEs necessitated romiplostim discontinuation. Two patients developed cytogenetic abnormalities, of whom one returned to normal karyotype at last follow‐up. High‐dose romiplostim is effective and well tolerated in the treatment of patients with AA refractory to IST.     

L‐Leucine improves the anaemia in models of Diamond Blackfan anaemia and the 5q‐ syndrome in a TP53‐independent way

Haploinsufficiency of ribosomal proteins (RPs) and upregulation of the tumour suppressor TP53 have been shown to be the common basis for the anaemia observed in Diamond Blackfan anaemia and 5q‐ myelodysplastic syndrome. We previously demonstrated that treatment with L‐Leucine resulted in a marked improvement in anaemia in disease models. To determine if the L‐Leucine effect was Tp53‐dependent, we used antisense MOs to rps19 and rps14 in zebrafish; expression of tp53 and its downstream target cdkn1a remained elevated following L‐leucine treatment. We confirmed this observation in human CD34+ cells. L‐Leucine thus alleviates anaemia in RP‐deficient cells in a TP53‐independent manner.     

Whole‐exome sequencing reveals the spectrum of gene mutations and the clonal evolution patterns in paediatric acute myeloid leukaemia

Acute myeloid leukaemia (AML) is a molecularly and clinically heterogeneous disease. Targeted sequencing efforts have identified several mutations with diagnostic and prognostic values in KIT, NPM1, CEBPA and FLT3 in both adult and paediatric AML. In addition, massively parallel sequencing enabled the discovery of recurrent mutations (i.e. IDH1/2 and DNMT3A) in adult AML. In this study, whole‐exome sequencing (WES) of 22 paediatric AML patients revealed mutations in components of the cohesin complex (RAD21 and SMC3), BCORL1 and ASXL2 in addition to previously known gene mutations. We also revealed intratumoural heterogeneities in many patients, implicating multiple clonal evolution events in the development of AML. Furthermore, targeted deep sequencing in 182 paediatric AML patients identified three major categories of recurrently mutated genes: cohesion complex genes [STAG2, RAD21 and SMC3 in 17 patients (8·3%)], epigenetic regulators [ASXL1/ASXL2 in 17 patients (8·3%), BCOR/BCORL1 in 7 patients (3·4%)] and signalling molecules. We also performed WES in four patients with relapsed AML. Relapsed AML evolved from one of the subclones at the initial phase and was accompanied by many additional mutations, including common driver mutations that were absent or existed only with lower allele frequency in the diagnostic samples, indicating a multistep process causing leukaemia recurrence.     

Prognostic value of paroxysmal nocturnal haemoglobinuria clone presence in aplastic anaemia patients treated with combined immunosuppression: results of two‐centre prospective study

Paroxysmal nocturnal haemoglobinuria (PNH) clones are frequently detected in patients with aplastic anaemia (AA). To evaluate the prognostic role of PNH clone presence we conducted a prospective study in 125 AA patients treated with combined immunosuppressive therapy (IST). Seventy‐four patients (59%) had a PNH clone (PNH+ patients) at diagnosis, with a median clone size of 0·60% in granulocytes and 0·15% in red blood cells. The response rate at 6 months was higher in PNH+ patients than that in PNH‐ patients, both after first‐ and second‐line IST: 68% vs. 45%, P = 0·0164 and 53% vs. 13%, P = 0·0502 respectively. Moreover, 42% of PNH+ patients achieved complete remission compared with only 16% of PNH‐ patients (P = 0·0029). In multivariate logistic regression analysis, PNH clone presence (odds ratio 2·56, P = 0·0180) and baseline absolute reticulocyte count (ARC) ≥30 × 10⁹/l (odds ratio 5·19, P = 0·0011) were independent predictors of response to treatment. Stratification according to PNH positivity and ARC ≥30 × 10⁹/l showed significant distinctions for cumulative incidence of response, overall and failure‐free survival. The results of this prospective study confirmed the favourable prognostic value of PNH clone presence in the setting of IST for AA.     

Impaired cellular and humoral immunity is a feature of Diamond-Blackfan anaemia; experience of 107 unselected cases in the United Kingdom

Diamond-Blackfan anaemia (DBA) is a rare bone marrow failure syndrome characterised by anaemia, congenital anomalies and cancer predisposition. Although infections are the second leading cause of mortality in non-transplanted patients, immune function is largely unexplored. We identified quantitative deficits in serum immunoglobulins and/or circulating T, natural killer and B lymphocytes in 59 of 107 unselected patients (55·1%) attending our centre over a 7-year period. Immune abnormalities were independent of ribosomal protein genotype and arose in both steroid-treated and steroid-untreated patients. In summary, these data highlight the high prevalence and spectrum of infections and immune defects in DBA.